The Friedreich ataxia (FRDA) is a frequent autosomal degenerative disease (1/30,000 live birth). It is characterized by spinocerebellar degeneration resulting in progressive limb and gait ataxia with lack of tendon reflexes in the legs and in pyramidal syndrome of the inferior limbs, and by hypertrophic cardiomyopathy. Geoffroy et al., Clinical description and roentgenologic evaluation of patients with Friedreich's ataxia, Can. J. Neurol. Sci. 3, 279-286 (1976); Harding, Friedreich's ataxia: a clinical and genetic study of 90 families with an analysis of early diagnostic criteria and intrafamilial clustering of clinical features, Brain, 104, 598-620 (1981). The disease gene has been mapped to chromosome 9q13 and encodes an ubiquitous 210-aminoacid protein, frataxin, targeted to the mitochondria. Chamberlain et al., Genetic homogeneity of the Friedreich ataxia locus on chromosome 9, Am. J. Human. Genet., 44, 518-521 (1989); Campuzano et al., Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion, Science, 271, 1423-1427 (1996); KoutnikovA et al., Studies of human, mouse and yeast homologue indicate a mitochondrial function for the frataxin, Nature Genet., 16, 345-351 (1997); Priller et al., Frataxin gene of Friedreich's ataxia is targeted to mitochondria, Ann. Neurol, 42, 265-269 (1997); Babcock et al., Regulation of mitochondrial iron accumulation by Yfh 1p, a putative homologue of fraxatin, Science, 276, 1709-1712 (1997); Foury et al., Deletion of the yeast homologue of the human gene associated with Friedreich's ataxia elicits iron accumulation in mitochondria, FEBS Lett., 411, 373-377 (1997); Wilson et al., Respiratory deficiency due to loss of mitochondrial DNA in yeast lacking the frataxin homologue, Nature Genet., 16, 352-357 (1997). FRDA is primarily caused by a GAA repeat expansion in the first intron of the fraxatin gene, which accounts for 98% of mutant alleles. Campuzano et al., Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion, Science, 271, 1423-1427 (1996). Yet, the tissue-specific expression of the disease remains unexplained and no animal model is presently available in FRDA.
The inventors have recently reported in Rotig et al., Aconitase and mitochondrial iron-sulphur protein defiency in Friedreich ataxia, Nature Genet., 17, 215-217 (1997) a deficient activity of the iron-sulphur (Fe-S) cluster containing proteins (ISP) in endomyocardical biopsies of FRDA patients, namely complexes I, II and III of the mitochondrial respiratory chain and aconitase, which cytosolic activity regulates cell iron homeostasis. Accordingly, ISPs have been shown to rapidly lose their catalytic activity in both FRDA patients and yeast strains carrying a deleted frataxin gene counterpart.
ISPs have been found to be remarkably sensitive to oxygen free radicals. Schoonen et al., Respiratory failure and stimulation of glycolysis in Chinese hamster ovary cells exposed to normobaric hyperoxia, J. Biol. Chem., 265, 1118-1124 (1990); Gardner et al., Aconitase is a sensitive and critical target of oxygen poisoning in cultured mammalian cells in rat lungs, Proc. Natl. Acad. Sci. USA, 91, 12248-12252 (1994); Li et al., Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutase, Nature Genet., 11, 376-381 (1996).
At the same time, iron has been found to be deposited in heart tissues of FRDA patients. Sanchez-Casis et al., Pathology of the heart in Friedreich's ataxia: review of the literature and report of one case, Can. J. Neurol. Sci., 3, 349-354 (1977).
Considering the remarkable sensitivity of ISPs to oxygen free radicals on the one hand and the reported iron deposit in heart tissues of FRDA patients on the other, the inventors have hypothesized that:
i) mitochondrial iron accumulation in FRDA is the consequence of the permanent activation of a mitochondrial iron import system, triggered by the decreased amount of frataxin, normally acting as a down regulator of mitochondrial iron uptake, and that PA1 ii) mitochondrial iron overload in FRDA would cause oxydative stress and an alteration of mitochondrial functions, through the iron-catalyzed Fenton chemistry. This posed the question of whether anti-oxidants would prevent the oxidative stress resulting from iron overload and alleviate the subsequent mitochondrial dysfunction.
Using an in vitro system, the inventors found that reduced (but not oxidized) iron was responsible for peroxidation of lipid membrane components and loss of membrane and soluble ISP activity, that antioxidants such as superoxide dismutase (SOD) and catalase were unable to prevent the iron-induced damages of the membrane components, and that reducing antioxidants such as ascorbate and glutathione were enhancing these damages.